No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Ultrastructural identification of the c-kit-expressing interstitial cells in the rat stomach: A comparison of control and Ws/Ws mutant rats
Abstract
Interstitial cells in the circular muscle layer of the stomach of the Ws/Ws mutant rat, which lacks c-kit-expressing cells, and its siblings have been studied by electron microscopy. In the sibling control rats, two types of interstitial cells are found lying in close association with nerve bundles. Cells of the first type are characterized by electron-dense cytoplasm containing abundant mitochondria, granular endoplasmic reticulum, and Golgi apparatus. Intermediate filaments are richly distributed throughout the perinuclear region and the cell processes. Caveolae, subsurface cisterns, and indistinct basal lamina are observed along the cell membrane. The most conspicuous feature of this cell type is the existence of many large gap junctions that interconnect with the same type of cell, smooth muscle cells, or cells of the second type. Cells of the second type show an ultrastructure similar to fibroblasts, viz., a well-developed Golgi apparatus and granular endoplasmic reticulum whose cisterns often show a dilated form and contain flocculent material. Unlike typical fibroblasts, however, cells of this type also form many gap junctions with cells of the first type and smooth muscle cells. Both types of cells are observed in close apposition to nerve varicosities. Since cells of the first type are absent in the Ws/Ws mutant rat, we concluded that they correspond to c-kit-expressing cells and to interstitial cells of Cajal.
... In addition we sought evidence for the hypothesis that all or some of these immature ICC might have features of ICC progenitor cells as deduced from studies on the murine stomach [21]. We were also interested in the relationship between potential progenitor cells based on immunohistochemistry and fibroblast-like ICC (FL-ICC) as identified by ultrastructural studies in the rat stomach [22]. ...
... The dominant interstitial cells associated with nerve structures in the wild-type and Ws/Ws colon were ICC, fibroblast-like ICC, fibroblasts, and macrophage-like cells. The term fibroblast-like ICC (FL-ICC) was used in accordance with the studies of Ishikawa and co-workers in the Ws/Ws rat stomach [22]. No obvious difference in the density of all interstitial cells combined surrounding Auerbach's plexus and the submuscular plexus were seen comparing wild-type and Ws/Ws colon. ...
... In the Ws/Ws colon, only a few ICC were present (Figure 7c). FL-ICC were present in much higher number with an electron-dense cytoplasm and all other characteristics of ICC except that they had conspicuous rough endoplasmic reticulum (rER) and were without caveolae as identified in the profiles studied, similar to the FL-ICC in the Ws stomach [22]. Typical examples are shown in figures 7d and 9c and 9d. ...
The colon of Ws/Ws mutant rats shows impairment of pacemaker activity and altered inhibitory neurotransmission. The present study set out to find structural correlates to these findings to resolve mechanisms. In the colon of Ws/Ws rats, interstitial cells of Cajal associated with Auerbach’s plexus (ICC-AP) were significantly decreased and ICC located at the submuscular plexus and intramuscular ICC were rarely observed based on immunohistochemistry and electron microscopy. Ultrastructural investigations revealed that there was no overall loss of all types of interstitial cells combined. Where loss of ICC was observed, a marked increase in fibroblast-like ICC (FL-ICC) was found at the level of AP. Immunoelectron microscopy proved FL-ICC to be c-Kit– but gap junction coupled to each other and to c-Kit+ ICC; they were associated with enteric nerves and occupied space normally occupied by ICC in the wild-type rat colon, suggesting them to be immature ICC. In addition, a marked increase in immunoreactivity for insulin-like growth factor 1 receptor (Igf1r) occurred, co-localized with CD34 but not with c-Kit. A significantly higher number of Igf1r+/CD34+ cells were found in Ws/Ws compared to wild-type rat colons. These CD34+/Igf1r+ cells in the Ws/Ws colon occupied the same space as FL-ICC. Hence we propose that a subset of immature ICC (FL-ICC) consists of adult progenitor cells. Immunohistochemistry revealed a reduction of neurons positive for neuronal nitric oxide synthase. The functional capabilities of the immature ICC and the regenerative capabilities of the adult progenitor cells need further study. The morphological features described here show that the loss of pacemaker activity is not associated with failure to develop a network of interstitial cells around AP but a failure to develop this network into fully functional pacemaker cells. The reduction in nitrergic innervation associated with the Ws mutation may be the result of a reduction in nitrergic neurons.
... GJs have been observed in almost every type of ICC (Komuro, 1999). They are particularly well developed in ICC thought to mediate nerve activity (Ward and Sanders, 2001a), such as ICC-DMP of the small intestine ( Fig. 11) and ICC-CM of the stomach (Fig. 12) (Ishikawa et al., 1997). ...
... GJs among ICC-LM were occasionally observed in the rat stomach (Mitsui and Komuro, 2002). Ishikawa et al., 1997.) b. ...
Interstitial cells of Cajal (ICC) are essential for the normal function of the digestive tract, both as pacemakers and as intermediates between nerves and smooth muscle cells. To perform their functions ICC must be electrically coupled both among themselves and to the muscle layers. This review focuses on the role gap junctions play in coupling ICC to ICC, providing a summary of the published literature as well as a critical appraisal of the data. Most of the experimental evidence for gap junction coupling of ICC networks is indirect, and consists of the ultrastructural observation of gap junctions. Dye coupling studies provide consistent support for the role of gap junctions among ICC of certain types. Physiological evidence in support of this role is scarce. The nature of ICC to smooth muscle coupling is even less certain.
... These studies suggest that there are macrophages with different morphologies and in various locations in the stomach as well. In a study of the ultrastructure of the circular muscle layer of antrum and corpus, Ishikawa et al. [81] described scattered nerve bundles and free cells (among them eosinophils and macrophages), which were occasionally observed in the interstitium around the nerve bundles. However, there is still a considerable lack of information on these macrophages in the stomach at both light and electron microscopic levels, especially with regard to their relation to ICC, and reports on serosal macrophages seem to be absent. ...
... In general, rats are considered to have more mast cells than mice, and Wistar rats to have more than Sprague-Dawley rats [83]. In the stomach Alcian-blue ϩ mast cells have been described in the submucosal layer of antrum and corpus, but very few were observed in the muscle layer [81]. In the muscularis externa of mouse small intestine, mast cells were extremely rare at both light and electron microscopical levels [4]. ...
Introduction
Identification of the cells
Cell distribution
Inflammation
Diabetes mellitus
Conclusions
Interstitial cells of Cajal (ICC) are recognized as pacemaker cells for gastrointestinal movement and are suggested to be mediators of neuromuscular transmission. Intestinal motility disturbances are often associated with a reduced number of ICC and/or ultrastructural damage, sometimes associated with immune cells. Macrophages and mast cells in the intestinal muscularis externa of rodents can be found in close spatial contact with ICC. Macrophages are a constant and regularly distributed cell population in the serosa and at the level of Auerbach’s plexus (AP). In human colon, ICC are in close contact with macrophages at the level of AP, suggesting functional interaction. It has therefore been proposed that ICC and macrophages interact. Macrophages and mast cells are considered to play important roles in the innate immune defence by producing pro‐inflammatory mediators during classical activation, which may in itself result in damage to the tissue. They also take part in alternative activation which is associated with anti‐inflammatory mediators, tissue remodelling and homeostasis, cancer, helminth infections and immunophenotype switch. ICC become damaged under various circumstances – surgical resection, possibly post‐operative ileus in rodents – where innate activation takes place, and in helminth infections – where alternative activation takes place. During alternative activation the muscularis macrophage can switch phenotype resulting in up‐regulation of F4/80 and the mannose receptor. In more chronic conditions such as Crohn’s disease and achalasia, ICC and mast cells develop close spatial contacts and piecemeal degranulation is possibly triggered.
... 20 Rats have IC-IM within the circular muscle layer of the stomach that run parallel to the circular muscle layer. 69 Short processes extend from these cells in various directions. These cells make frequent, large gap junctions with each other and with neighbouring smooth muscle cells. ...
... The circular muscle of W S /W S animals is not distinguishable from that of wildtype animals; however, the dark cells identi®ed as IC-IM were not found. 69 Cells expressing c-kit were also not observed in the myenteric regions of the stomach of W S /W S rats, re¯ecting loss of this class of ICC as well. 20 The number of c-kit expressing cells was also greatly reduced in the myenteric (IC-MY) plexus region of the ileum and in the myenteric plexus (IC-MY) and submucosal surface of the circular muscle (IC-SM) in the colon. ...
Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and these cells also mediate or transduce inputs from the enteric nervous system. Different classes of ICC are involved in pacemaking and neurotransmission. ICC express specific ionic conductances that make them unique in their ability to generate and propagate slow waves in GI muscles or transduce neural inputs. Much of what we know about the function of ICC comes from developmental studies that were made possible by the discoveries that ICC express c-kit and proper development of ICC depends upon signalling via the Kit receptor pathway. Manipulating Kit signalling with reagents to block the receptor or downstream signalling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies into the specific functions of the different classes of ICC in several regions of the GI tract. Kit is also a surface antigen that can be used to conveniently label ICC in GI muscles. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells in the gut wall, and the loss of ICC in some clinical disorders. Preparations made devoid of ICC have also allowed analysis of the consequences of losing specific classes of ICC on GI motility. This review describes recent advances in our knowledge about the development and plasticity of ICC and how developmental studies have contributed to our understanding of the functions of ICC. We have reviewed the clinical literature and discussed how loss or defects in ICC affect GI motor function.
... The distribution of ICC was indistinguishable from that of wild-type animals (12,13,15,18). The ultrastructure of gastric IC-MY and IC-IM was examined by transmission electron microsopy, and these cells were identified by commonly recognized features (13,(15)(16)(17)20,31,32) (Fig. 3A and C). IC-MY and IC-IM were usually closely associated with enteric ganglia or nerve fibers within the muscle layers (13,(15)(16)(17)20,28,(30)(31)(32) (Fig. 3A and C). ...
... The ultrastructure of gastric IC-MY and IC-IM was examined by transmission electron microsopy, and these cells were identified by commonly recognized features (13,(15)(16)(17)20,31,32) (Fig. 3A and C). IC-MY and IC-IM were usually closely associated with enteric ganglia or nerve fibers within the muscle layers (13,(15)(16)(17)20,28,(30)(31)(32) (Fig. 3A and C). ...
Patients with long-standing diabetes commonly suffer from gastric neuromuscular dysfunction (gastropathy) causing symptoms ranging from postprandial bloating to recurrent vomiting. Autonomic neuropathy is generally believed to be responsible for diabetic gastropathy and the underlying impairments in gastric emptying (gastroparesis) and receptive relaxation, but the specific mechanisms have not been elucidated. Recently, it has been recognized that interstitial cells of Cajal generate electrical pacemaker activity and mediate motor neurotransmission in the stomach. Loss or defects in interstitial cells could contribute to the development of diabetic gastroparesis. Gastric motility was characterized in spontaneously diabetic NOD/LtJ mice by measuring gastric emptying and by monitoring spontaneous and induced electrical activity in circular smooth muscle cells. Interstitial cells of Cajal were studied by Kit immunofluorescence and transmission electron microscopy. Diabetic mice developed delayed gastric emptying, impaired electrical pacemaking, and reduced motor neurotransmission. Interstitial cells of Cajal were greatly reduced in the distal stomach, and the normally close associations between these cells and enteric nerve terminals were infrequent. Our observations suggest that damage to interstitial cells of Cajal may play a key role in the pathogenesis of diabetic gastropathy.
... SMCs are electrically coupled to 2 types of interstitial cells. Interstitial cells of Cajal (ICC) and fibroblast-like cells (PDGFRa þ cells) are coupled to SMCs by gap junctions, 18,19 forming an electrical syncytium known as the SIP (abbreviation for SMCs, ICC and PDGFRa þ cells) syncytium. 20 The syncytial nature of gastric muscles means that electrical responses that develop in interstitial cells can conduct to SMCs and regulate the excitability and motor activity of SMCs. ...
Gastroparesis is characterized by symptoms suggestive of and objective evidence of delayed gastric emptying in the absence of mechanical obstruction. This review addresses the normal emptying of solids and liquids from the stomach and details the myogenic and neuromuscular control mechanisms including the specialized function of the pyloric sphincter that result in normal emptying, based predominantly on animal research. A clear understanding of fundamental mechanisms is necessary to comprehend derangements leading to gastroparesis, and additional research on human gastric muscles is needed. The section on pathophysiology of gastroparesis considers neuromuscular diseases that affect non-sphincteric gastric muscle, disorders of the extrinsic neural control and pyloric dysfunction that lead to gastroparesis. The potential cellular basis for gastroparesis is attributed to the effects of oxidative stress and inflammation, with increased pro-inflammatory and decreased resident macrophages, as observed in full-thickness biopsies from patients with gastroparesis. Predominant diagnostic tests involving measurements of gastric emptying, the use of a functional luminal imaging probe and high-resolution antral duodenal manometry in characterizing the abnormal motor functions at the gastroduodenal junction are discussed. Management is based on supporting nutrition, dietary interventions including the physical reduction in particle size of solid foods, pharmacological agents including prokinetics and anti-emetics, and interventions such as gastric electrical stimulation and pyloromyotomy. These are briefly discussed, and comment is added on the potential for individualized treatments in the future, based on optimal gastric emptying measurement and objective documentation of the underlying pathophysiology causing the gastroparesis.
... These gastric layers are evident upon hemoxylin and eosin staining, alongside the other layers of the stomach; submucosa, and muscularis externa (Ajayi & Olaleye, 2015;Cha, 2007;Feldman & Wolfe, 2014). The submucosal layer is host to mast cells, which are also sparsely distributed in the muscularis mucosa and can be detected with the toluidine blue stain (Ishikawa et al., 1997). Mast cell degranulation in response to allergies elevates gastric motility and the release of histamin and other mediators (Beyak et al., 2006). ...
The interaction between ingested xenobiotics and the gastrointestinal epithelium influences the possibility of gut epithelial cytotoxicity and systemic toxicity. Potassium bromate (KBrO3) has been shown to perturb the central nervous system and it may be carcinogenic, albeit it is used as a food additive. This highlights the need to understand KBrO3’s effect on the stomach epithelium. Here, we report the cytotoxic potential of KBrO3 in an ulcerated stomach, as well as possible cytoprotection by the polyphenol ‒ protocatechuic acid. Potassium bromate (12.5 mg/kg) and protocatechuic acid (120 mg/kg) were administered orally while omeprazole (20 mg/kg) was used as standard. Potassium bromate exacerbated gastric ulcers, increased malonaldehyde levels, catalase, and sodium pump activities, but reduced nitric oxide levels. Potassium bromate further increased mast cell count in the muscularis mucosa, while inducing chronic inflammation and moderate angiogenesis in the gastric mucosa. Our results delineate KBrO3‐induced gastric epithelial cytotoxicity that is ameliorated by protocatechuic acid.
Practical applications
Potassium bromate is a known food additive in the baking, brewing, and cheese‐making process. Conversely, protocatechuic acid (3,4‐dihydroxybenzoic acid) is the polyphenolic content of plants like Hibiscus sabdariffa L that are commonly consumed as herbal drink, food, spices, and used in folk medicine. This study reports the cytoprotective effect of protocatechuic acid against gastric mucosa ulceration that has been aggravated by potassium bromate.
... 众所周知，胃肠道运动除受外来的交感和副交 感神经支配外，还接受肠神经系统 (enteric nervous system, ENS) 的调节。构成 ENS 的主要成分是分布 在消化道纵行肌和环形之间的肌间神经丛 (myenteric plexus)，也称为欧氏神经丛 (Auerbach's plexus)，其 中包含传入神经元、中间神经元和传出神经元等， 它们负责调节胃肠道的运动，保证正常的消化和吸 收功能 [1][2][3][4] 。肠神经对消化道平滑肌运动调节包括 兴奋性和抑制性调节，兴奋性调节主要是由兴奋性 运动神经元通过释放兴奋性神经递质，如乙酰胆碱、 P 物质等增加平滑肌细胞的兴奋性，而抑制性调节 则由抑制性运动神经元通过释放抑制性神经递质， 如 NO、ATP、ADP 和 β-烟 酰 胺 腺 嘌 呤 二 核 苷 酸 (β-nicotinamide adenine dinucleotide, β-NAD) 等嘌呤 (purines) 能递质降低平滑肌细胞的兴奋性。在神经 信息传递过程方面，消化道平滑肌与骨骼肌机制完 全不同，神经对骨骼肌的支配是通过运动单位的精 细调节，即运动神经末梢与骨骼肌肌纤维之间通过 神经 -肌肉接头把神经信息传递给骨骼肌。而支配 消化道平滑肌的神经末梢则形成许多膨大的曲张体 (varicosity)，其中包含有神经递质，但是在该结构 当中没有发现类似骨骼肌那样的神经 -肌肉接头， 因此研究者们认为曲张体释放的神经递质是以扩散 的方式到达平滑肌发挥作用的。但随着消化道两种 间质细胞即 Cajal 间质细胞 (interstitial cell of Cajal, ICC) 和血小板衍生生长因子受体 α 阳性 (plateletderived growth factor receptor α positive, PDGFRα + ) 细胞分布和功能的深入研究，人们对于神经信息在 肠神经和平滑肌之间的传递有了新的认识 [5] 。研究 显示，在电镜下，肠神经末梢的曲张体周围紧紧 围绕着两种间质细胞 --ICC 和 PDGFRα + 细胞 [6] ， 而且这两种间质细胞分别通过缝隙连接与平滑肌细 胞形成电耦联 (electric coupling)，这种平滑肌、ICC 和 PDGFRα + 细胞组成的结构被称为 SIP 合胞体 (SIP syncytium) [ [20] 。Baker 等 [21] 在小鼠的结肠平滑肌也做了类似的工作，即通过 ...
Under physiological conditions, the motility of smooth muscle in digestive tract is mainly regulated by enteric nervous system (ENS). However, how neural signal is transmitted to smooth muscle is not fully understood. Autonomic nerve endings in the smooth muscle layer form large number of varicosities which contain neurotransmitters. It was considered that nerve pulses arriving at the varicosities may cause the release of neurotransmitters, which may diffuse to the smooth muscle cells to induce contractile or relaxant responses. Over the past decade, a new understanding of the neurotransmission between ENS and smooth muscle has emerged, which emphasizes the role of a functional syncytium consisting of the interstitial cells of Cajal (ICC), the platelet-derived growth factor receptor α positive (PDGFRα+) cells and the smooth muscle cells. Within the syncytium, purine neurotransmitters bind to P2Y1 receptors on PDGFRα+ cells, activating small-conductance calcium activated potassium channel (SK3) to hyperpolarize PDGFRα+ cells, and thus hyperpolarize smooth muscle cells through gap junction, resulting in relaxation of smooth muscle. In this paper, we review the research progress in the field of inhibitory purinergic neurotransmission in the gastrointestinal tract.
... In the IAS, synchronisation of Type II cells occurs in spite of the fact that these cells do not form a network with one another. However, ICC-IM are electrically coupled to SMCs via gap junctions 11,50,51 . Thus, SMCs may act as a bridge in the propagation of SW activity between ICC-IM. ...
The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca²⁺ transients in mice expressing a genetically-encoded Ca²⁺-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca²⁺ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca²⁺ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca²⁺ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.
... Therefore, the ICC-SM of the stomach can also generate slow waves. The ultrastructure of this type of cell in the rat stomach 43 , in which ICC-SM were characterized as having cytoplasmic organelles, including abundant mitochondria, rough endoplasmic reticula, and rich intermediate filaments, as well as numerous caveolae and a discontinuous basal lamina. In the present study, we observed the morphology and distribution of ICC-SM are consistent with their studies, so here we hypothesized that the ICC-SM can also generate a slow wave in goat abomasum. ...
The interstitial cells of Cajal (ICCs) are regarded as pacemakers and are involved in neurotransmission in the gastrointestinal tract (GIT) of animals. However, limited information is available about the existence of ICCs within the GIT of ruminants. In this study, we investigated the ultrastructural characteristics and distribution of ICCs in goat abomasum using transmission electron microscopy and c-kit immunohistochemistry. Two different kinds of c-kit immunoreactive cells were observed in the abomasum. The first was identified as ICCs, which appeared to be multipolar or bipolar in shape, with some processes. These c-kit immunoreactive cells were deposited in the submucosal layer, myenteric plexus between the circular and longitudinal muscle layers, and within the longitudinal and circular muscle layers of the abomasum. The second type of cell was round in shape and was identified as mast cells, which were located in the submucosal layer as well as in the lamina propria. Ultrastructurally, ICCs were also observed as stellate or spindle-shaped cells, which were consistent in shape with our c-kit immunoreactive cells. In the cytoplasm of ICCs, numerous mitochondria, rough endoplasmic reticulum, and caveolae were detected. ICCs were located in the myenteric plexus between the longitudinal and circular muscle layers (ICC-MY), with the longitudinal and circular muscle layer was replaced as “intramuscular layers” (ICC-IM), and in the submucosal layer (ICC-SM). In addition, we found ICCs surrounding nerve fibers and smooth muscle cells, where they formed heterocellular junctions in the form of close membrane associations or gap junctions and homocellular junctions among the processes of the ICCs. In the current study, we provide the first complete characterization of ICCs within the goat abomasum and propose that ICCs might have a key role in producing contractions in the ruminant stomach for proper absorption of nutrients.
... Since that time ICC were detected in many other tissues, always in the vicinity of smooth muscle cells. ICC were identified in alimentary tract of many species, i.e. mice [3], rats [4], guinea pigs [5]. In humans ICC were found in the wall of alimentary tract [6], pancreas [7], in the muscle of atria and ventricles [8,9,10], vagina [11], mammary gland [12,13], oviduct [14], ductus deferens [15], urinary tract [16,17,18] [26,28]. ...
This paper reviews the distribution of interstitial cells of Cajal (ICC) in the human gastrointestinal (GI) tract, based on ultrastructural and immunohistochemical evidence. The distribution and morphology of ICC at each level of the normal GI tracts is addressed from the perspective of their functional significance. Alterations of ICC reported in as well as in gastrointestinal stromal tumors are reviewed, with emphasis on the place of ICC in the pathophysiology of disease.
... 5 There are at least 2 distinct classes of ICC in the stomach. ICC-MY lie in the plane of the myenteric plexus between the circular and longitudinal muscle layers, 7,8 and intramuscular ICC (ICC-IM) lie within muscle bundles throughout most of the stomach. 9 However, examination of antral longitudinal muscles of some species shows few ICC-IM in this region. ...
Gastric peristalsis begins in the orad corpus and propagates to the pylorus. Directionality of peristalsis depends upon orderly generation and propagation of electrical slow waves and a frequency gradient between proximal and distal pacemakers. We sought to understand how chronotropic agonists affect coupling between corpus and antrum.
Electrophysiological and imaging techniques were used to investigate regulation of gastric slow wave frequency by muscarinic agonists in mice. We also investigated the expression and role of cholinesterases in regulating slow wave frequency and motor patterns in the stomach.
Both acetycholinesterase (Ache) and butyrylcholine esterase (Bche) are expressed in gastric muscles and AChE is localized to var-icose processes of motor neurons. Inhibition of AChE in the absence of stimulation increased slow wave frequency in corpus and throughout muscle strips containing corpus and antrum. CCh caused depolarization and increased slow wave frequency. Stimulation of cholinergic neurons increased slow wave frequency but did not cause depolarization. Neostigmine (1 μM) in-creased slow wave frequency, but uncoupling between corpus and antrum was not detected. Motility mapping of contractile activity in gastric muscles showed similar effects of enteric nerve stimulation on the frequency and propagation of slow waves, but neostigmine (> 1 μM) caused aberrant contractile frequency and propagation and ectopic pacemaking.
Our data show that slow wave uncoupling is difficult to assess with electrical recording from a single or double sites and sug-gest that efficient metabolism of ACh released from motor neurons is an extremely important regulator of slow wave frequency and propagation and gastric motility patterns.
... 22 In addition to mice, Kit mutant rats (Ws/Ws) have also been shown to have lesions in ICC-MY in the small intestine 97,118 and ICC-IM in the stomach. 119 Intestinal motility was shown to be disrupted in these rats. 120 Smooth muscle tissues and cells appear to be unaffected in W mutants and are capable of producing Ca 2+ action potentials, responses to agonists and contractile responses. ...
Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha (PDGFRα+) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα+ cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.
... We have demonstrated the presence of c-kit positive cells in bovine forestomachs at all developmental stages studied. Earlier studies of mice (Maeda et al. 1992), guinea pigs (Komuro et al. 1996), rats (Ishikawa et al. 1997), humans (R ø mert and Mikkelsen 1998), dogs (Horiguchi et al. 2001), cats (Morini et al. 2004) and horses (Hudson et al. 1999) among other species have shown that c-kit immunostaining, together with cellular morphology and localization, enabled identifi cation of these cells as ICC (Faussone-Pellegrini and Thuneberg 1999). Vimentin KHC was positive in the same areas as c-kit positive cells, although the vimentin staining was observed over a larger area, because fi broblasts contain vimentin intermediate fi laments (Torihashi et al. 1993). ...
The interstitial cells of Cajal (ICC) have been reported to regulate gastrointestinal motility. We investigated the distribution and the morphological and morphometric characteristics of the immunohistochemical reaction against c-kit in the forestomachs of fetal, newborn and adult cows. The anti-c-kit reaction revealed different populations of ICC among age groups and organs. ICC were more numerous and smaller in fetuses. Larger ICC were identified in newborns, except for those in the rumen. During the earliest stages of development, ICC were abundant in the inner layer of the muscularis and were consistently associated with this layer. In all samples, ICC were found in the outer layer of the tunica muscularis. ICC were found between the two muscle layers in the omasum at all ages; however, they were identified only in the rumen of the adult. Our study demonstrated that ICC are present in the forestomach of bovines.
... These cells, referred to as fibroblast-like cells, are found near terminals of motor neurons and form gap junctions with SMCs. [130][131][132] Fibroblast-like cells express small conductance Ca 2+ -activated K + channels, SK3 (encoded by KCNN3), [133][134][135][136] which might be activated in purinergic inhibitory responses. These fibroblast-like cells are labelled with antibodies to PDGFRα, and PDGFRα + cells express SK3 channels and P2Y1 receptors. ...
Gastrointestinal motility results from coordinated contractions of the tunica muscularis, the muscular layers of the alimentary canal. Throughout most of the gastrointestinal tract, smooth muscles are organized into two layers of circularly or longitudinally oriented muscle bundles. Smooth muscle cells form electrical and mechanical junctions between cells that facilitate coordination of contractions. Excitation-contraction coupling occurs by Ca(2+) entry via ion channels in the plasma membrane, leading to a rise in intracellular Ca(2+). Ca(2+) binding to calmodulin activates myosin light chain kinase; subsequent phosphorylation of myosin initiates cross-bridge cycling. Myosin phosphatase dephosphorylates myosin to relax muscles, and a process known as Ca(2+) sensitization regulates the activity of the phosphatase. Gastrointestinal smooth muscles are 'autonomous' and generate spontaneous electrical activity (slow waves) that does not depend upon input from nerves. Intrinsic pacemaker activity comes from interstitial cells of Cajal, which are electrically coupled to smooth muscle cells. Patterns of contractile activity in gastrointestinal muscles are determined by inputs from enteric motor neurons that innervate smooth muscle cells and interstitial cells. Here we provide an overview of the cells and mechanisms that generate smooth muscle contractile behaviour and gastrointestinal motility.
... Ligand-binding is followed by receptor dimerization, autophosphorylation and activation of downward signaling pathways such as MAPK, JAK/STAT and PI3K/AKT pathways (Duensing et al., 2004;Mol et al., 2003). Kit was found to be expressed by a variety of cell types including the interstitial cells of Cajal, mast cells, haemopoietic progenitor cells or melanocytes (Natali et al., 1992;Nocka et al., 1989;Turner et al., 1992;Ishikawa et al., 1997), and its dysregulation has been associated with the pathogenesis of various different human malignancies (Duensing & Duensing, 2010;Heinrich et al., 2002;Woodman & Davies, 2010). In glioma, about 75% of the tumors were reported to express Kit (Cetin et al., 2005). ...
... How activation of ion channels in PDGFRα + cells can influence the behaviour of colonic muscles should also be discussed. Cells previously identified as 'fibroblast-like' cells in GI muscles form gap junctions with surrounding smooth muscle cells as shown by ultrastructural studies [14,29]. Gap junctions provide electrical coupling between cells, such that induction of a K + current (i.e. ...
An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express platelet-derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon. Fibroblast-like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet-derived growth factor receptor α(+) cells are distinct from c-Kit(+) interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet-derived growth factor receptor α(+) cells express small conductance Ca(2+)-activated K(+) channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα(+) cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα(+) cells in the human GI tract in health and disease.
... Another cell type, referred to as 'fibroblast-like cells' (FLCs; Ishikawa et al. 1997;Horiguchi & Komuro, 2000;J Physiol 588.23 Figure 2. Relationship of motor nerve varicosities to interstitial cells and smooth muscle cells ICC of the circular muscle layer (ICC-IM) and fibroblast-like cells (PDGFRα + cells) are commonly found closely associated with nerve bundles (NB), as in this section from rat stomach (A; scale bar is 0.5 μm; reproduced with kind permission from Springer Science+Business Media: Mitsui & Komuro, 2002, Cell & Tissue Res 309, 219-227). ...
Control of gastrointestinal (GI) movements by enteric motoneurons is critical for orderly processing of food, absorption of nutrients and elimination of wastes. Work over the past several years has suggested that motor neurotransmission is more complicated than simple release of transmitter from nerve terminals and binding of receptors on smooth muscle cells. In fact the 'neuro-effector' junction in the tunica muscularis might consist of synaptic-like connectivity with specialized cells, and contributions from multiple cell types in integrated post-junctional responses. Interstitial cells of Cajal (ICC) were proposed as potential mediators in motor neurotransmission based on reduced post-junctional responses observed in W mutants that have reduced populations of ICC. More recent studies on W mutants have contradicted the original findings, and suggested that ICC may not be significant players in motor neurotransmission. This review examines the evidence for and against the role of ICC in motor neurotransmission and outlines areas for additional investigation that would help further resolve this controversy.
... Through different signal transduction pathways and tissue-specific regulation, cKIT affects development and maintenance of several cell systems including hematopoiesis [4][5][6][7]. Upon binding of Kit ligand (KL), Kit forms a homodimer, autophosphorylates tyrosine residues and induces diverse signal transduction cascades involving SHP-1, SHP-2, SRC kinase family and phosphoinositide-3 kinase (PI3-kinase) [8][9][10][11]. Activating mutations of the cKIT gene have been reported in between 12.8 and 46.1% of adult CBFL and in 12% of pediatric acute promyelocytic leukaemia (APL) [12][13][14]. ...
The Wnt-signaling pathway plays a critical role in directing cell fate during embryogenesis and also in the pathogenesis of cancer. In leukemia, it is well described that activating internal tandem duplications (ITD) mutations in receptor tyrosine kinases like cKit or Flt3 confer to the pathogenesis of cancer. Here, we analyzed whether Wnt-signaling plays a role in cKit-ITD mediated transformation. Stably transfected 32D cells with cKit-ITD cells had higher beta-Catenin protein levels compared to the cKit-WT. Analysis of beta-Catenin mRNA and protein levels revealed that beta-Catenin was regulated at post-transcriptional level in cKit-ITD as well as Flt3-ITD compared to the wildtype. Signaling analyses revealed higher-phosphorylation of GSK3beta by oncogenic cKit-ITD. Moreover, activation of Wnt signaling was confirmed by constitutive activation of c-myc luciferase by cKit-ITD cells. Importantly, using dominant negative TCF4, we show that activation of Wnt signaling plays an important role in cKit mediated transformation of myeloid cells. Application of specific receptor tyrosine kinase inhibitors for Flt3 or cKit result in a decrease of beta-Catenin that underwent with a decrease of GSK3beta phosphorylation, suggesting an indirect mechanism of beta-Catenin regulation by oncogenic receptor tyrosine kinases in both ITD mutations. Our study shows the importance of activation of Wnt signaling in leukemia and suggests as attractive target for future therapeutical approaches.
... Two populations of morphologically distinct immunoreactive c-kit cells have been identified in the digestive tract of mammals and humans, namely the mastocytes (Vliagoftis et al. 1997) and the interstitial cells (Sanders 1996). C-kit immunoreactive interstitial cells (c-kit ICs) have been found to exist in various species, such as mice (Maeda et al. 1992; Huizinga et al. 1995; Torihashi et al. 1997; Ward et al. 1997), rats (Isozaki et al. 1995; Ishikawa et al. 1997; Horiguchi and Komuro 1998), and guinea pigs (Komuro and Zhou 1996; Burns et al. 1997; Seki et al. 1998), as well as humans (Horie et al. 1993; Matsuda et al. 1993; Rumessen 1994; Vanderwinden et al. 1996a,b; Hagger et al. 1997 Hagger et al. , 1998 Horisawa et al. 1998; Romert and Mikkelsen 1998; Torihashi et al. 1999; Wester et al. 1999 ). All in all, these studies have shown that the distribution and morphology of the c-kit ICs vary from one species to another, as well as from one region of the digestive tract to another, within a given species. ...
C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".
Propulsion of contents in the gastrointestinal tract requires coordinated functions of the extrinsic nerves to the gut from the brain and spinal cord, as well as the neuromuscular apparatus within the gut. The latter includes excitatory and inhibitory neurons, pacemaker cells such as the interstitial cells of Cajal and fibroblast-like cells, and smooth muscle cells. Coordination between these extrinsic and enteric neurons results in propulsive functions which include peristaltic reflexes, migrating motor complexes in the small intestine which serve as the housekeeper propelling to the colon the residual content after digestion, and mass movements in the colon which lead to defecation.
Purpose of review:
Gastroparesis is a chronic disorder characterized by a constellation of foregut symptoms, including postprandial nausea, vomiting, distension, epigastric pain, and regurgitation in the absence of gastric outlet obstruction. Despite considerable research over the past decades, there remains to be only nominal understanding of disease classification, diagnostic criteria, pathogenesis, and preferred therapy.
Recent findings:
We critically reassess current approaches for disease identification and stratification, theories of causation, and treatment for gastroparesis. Gastric scintigraphy, long considered a diagnostic standard, has been re-evaluated in light of evidence showing low sensitivity, whereas newer testing modalities are incompletely validated. Present concepts of pathogenesis do not provide a unified model linking biological impairments with clinical manifestations, whereas available pharmacological and anatomical treatments lack explicit selection criteria or evidence for sustained effectiveness. We propose a disease model that embodies the re-programming of distributed neuro-immune interactions in the gastric wall by inflammatory perturbants. These interactions, combined with effects on the foregut hormonal milieu and brain-gut axis, are postulated to generate the syndromic attributes characteristically linked with gastroparesis. Research linking models of immunopathogenesis with diagnostic and therapeutic paradigms will lead to reclassifications of gastroparesis that guide future trials and technological developments.
Key points:
• The term gastroparesis embodies a heterogenous array of symptoms and clinical findings based on a complex assimilation of afferent and efferent mechanisms, gastrointestinal locations, and pathologies. • There currently exists no single test or group of tests with sufficient capacity to be termed a definitional standard for gastroparesis. • Present research regarding pathogenesis suggests the importance of immune regulation of intrinsic oscillatory activity involving myenteric nerves, interstitial cells of Cajal, and smooth muscle cells. • Prokinetic pharmaceuticals remain the mainstay of management, although novel treatments are being studied that are directed to alternative muscle/nerve receptors, electromodulation of the brain-gut axis, and anatomical (endoscopic, surgical) interventions.
Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit+ (interstitial cells of Cajal or ICC), and PDGFRα+ cells (formerly “fibroblast-like” cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca2+ release from the endoplasmic reticulum (ER) and activation of Ca2+-activated ion channels in the plasma membrane (PM). ICC express Ca2+-activated Cl− channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα+ cells express Ca2+-activated K+ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca2+ transients in ICC and PDGFRα+ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca2+ conductances that organize Ca2+ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca2+ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca2+ dynamics in these cells in GI motility disorders.
STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.
PURPOSE: Activation of the KIT tyrosine kinase by somatic mutation has been documented in a number of human malignancies, including gastrointestinal stromal tumor (GIST), seminoma, acute myelogenous leukemia (AML), and mastocytosis. In addition, paracrine or autocrine activation of this kinase has been postulated in numerous other malignancies, including small-cell lung cancer and ovarian cancer. In this review, we discuss the rationale for and development of KIT tyrosine kinase inhibitors for the treatment of human malignancies.
MATERIALS AND METHODS: Studies were identified through a MEDLINE search, review of bibliographies of relevant articles, and review of abstracts from national meetings.
RESULTS: Four tyrosine kinase inhibitors that have activity against KIT are currently being used in clinical trials, and one, STI571, has recently been approved by the United States Food and Drug Administration for treating patients with chronic myelogenous leukemia. The role of KIT inhibitors in treating KIT-positive malignancies is reviewed.
CONCLUSION: Targeted therapy to inhibit the kinase activity of KIT is a rational approach to the treatment of KIT-positive malignancies. Two key factors are the potency of a given inhibitor and the relative contribution of KIT activation to the growth of the tumor. Given our current understanding of KIT activity in human malignancy, the best candidate diseases for treatment with KIT inhibitors are GIST, mastocytosis, seminoma and possibly some cases of AML. Additionally, KIT inhibitors may play an adjunctive role in diseases such as small-cell lung cancer, in which KIT activation is secondary to ligand binding rather than an acquired mutation.
AIM: To investigate the impact of chronic water immersion-restraint stress on the ultrastructure of interstitial cells of Cajal (ICC) in the gastric antrum of rats. METHODS: Forty-eight male Sprague-Dawley rats were randomly and equally divided into six groups: three experimental groups and three matched control groups. The three experimental groups underwent water immersion-restraint stress for one hour daily for 3, 7 and 14 days, respectively, while the three control groups were allowed free access to food and water. On days 4, 15 and 28, the rats in both the experimental and control groups were sacrificed. Two pieces of antrum tissues were taken from each rat and fixed in 3% glutaraldehyde for electron microscopic examination. RESULTS: ICC were normal in all the control groups but showed visible injuries in the three experimental groups. With the prolongation of the stress, the morphological damage became more evident in ICC, especially in inter-muscular ICC (ICC-MY) and intramuscular ICC (ICC-IM). Major ultrastructural changes in ICC included reduced number of gap junctions and organelles. CONCLUSION: Chronic water immersion-restraint stress can lead to ultrastructural damage in ICC in the gastric antrum of rats.
AIM: To investigate the alterations in the ultrastructure of interstitial cells of Cajal (ICC) in the gastric antrum of rats undergoing chronic water immersion-restraint stress. METHODS: Forty-eight male Sprague-Dawley rats were randomly and equally divided into six groups: three experimental groups and three matched control groups. The three experimental groups underwent water immersion-restraint stress for one hour daily for 3, 7 and 28 days, respectively, while the three control groups were allowed free access to food and water. On days 4, 8 and 29, the rats in both the experimental and control groups were sacrificed. Two pieces of antrum tissues were taken from each of three rats in each group and fixed in 3% glutaraldehyde for electron microscopic examination. The severity of injury was then scored. RESULTS: Compared to the control groups, the ICC in the gastric antrum of rats in the experimental groups showed widened perinuclear space, discontinuous basement membrane, cytoplasmic dissolution and vacuolation, decreased number of gap junctions, mitochondrial swelling and vacuolation, dilated endoplasmic reticulum, decreased amount of rough endoplasmic reticulum, and nuclear abnormality. With the prolongation of stress duration, the ultrastructural injury to ICC was aggravated, particularly prominent in cytoplasmic dissolution and vacuolation and the decrease in the amount of rough endoplasmic reticulum. CONCLUSION: Chronic water immersion-restraint stress can induce ultrastructural injury to ICC in the rat gastric antrum.
The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP dependent protein kinase I (cGKI) were examined in cryostat sections with dual labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC) and smooth muscle cells (SMC). Gene expression levels were determined with qPCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+) and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were: PDGFRα(+) cells>ICC>SMC. In contrast, cGKI gene expression sequence was SMC=ICC>PDGFRα(+) cells while cGKI protein expression sequence was neurons>SMC>ICC=PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-Br-cGMP was greatly reduced in cGKI(-/-) mice while responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5Hz, 60s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. L-NNA abolished responses during the first 20-30s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.
Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.
Interstitial cells of Cajal (ICC) are responsible for generating electrical slow waves in the gastrointestinal (GI) tract. Slow waves regulate the frequency of contractions of the tunica muscularis, and therefore ICC are critical for normal motility in the small intestine. ICC express Kit, the gene product of c-kit, a protooncogene that encodes a receptor tyrosine kinase. Physiological evidence demonstrating that ICC are pacemakers has come from experiments on W-mutant mice which have few Kit-positive cells at the level of the myenteric plexus (IC-MY) and also lack electrical slow waves. In the past identification of ICC required the use of electron microscopy, however the discovery that ICC express Kit has facilitated studies of the distribution of ICC in several species. Immunoelectron microscopy to relate ultrastructure to Kit expression has only been performed in a limited number of studies of mice. We examined the ultrastructure of Kit-expressing cells in the rat using immunoelectron microscopy and an anti-Kit antibody. We compared the presence and appearance of Kit-expressing ICC in wildtype and Ws/Ws rats, which carry a mutation in the white spotting locus and have a phenotype similar to W/W
V
mutant mice. Kit-expressing cells could be detected in the myenteric plexus (MY) and deep muscular plexus (DMP) regions of the small intestine of wildtype animals. In Ws/Ws rats, Kit-expressing cells were not observed in the region of MY, but were observed in the DMP. The density of Kit-positive cells in the DMP of Ws/Ws rats was similar to those in wildtype rats. Electron microscopy showed that Kit-expressing cells at the level of the MY of the rat had similar ultrastructural features as IC-MY in wildtype mice. IC-DMP in the rat of both wildtype and Ws/Ws mutants were similar in structure to IC-DMP of the mouse. We conclude that wildtype rats have IC-MY and IC-DMP in the tunica muscularis of the jejunum. ICC express Kit-like immunoreactivity (Kit-LI) in the rat as in the mouse. IC-MY are absent in the small intestine of Ws/Ws rats, and this corresponds to the lack of Kit-labeling in this region. Ws/Ws rats, however, possess IC-DMP with normal ultrastructural features and Kit-LI. The absence of IC-MY of Ws/Ws rats is likely to account for the abnormal contractile activity of the GI tract observed in these mutants. The present study suggests that Ws/Ws rats could provide an interesting model to investigate the physiological significance of pacemaker activity because they manifest a defect in IC-MY.
In vitro studies on pacemaker-deficient W-mutants have revealed a disappearance of rhythmic contraction in their gastrointestinal tracts. Their contractile force has not been diminished, however. In contrast, W-mutants often present dysmoility-like symptoms with distension of the gastrointestinal tract in vivo. Gastrointestinal motility of W-mutant rats was examined in vivo by an extraluminal strain-gauge force transducer method. We examined a normal gastrointestinal motor pattern in the rats with two distinct motor phases, digestive and interdigestive. Moreover, we detected a failure to form an interdigestive contractile complex in pacemaker-deficient rats. The interdigestive motor activity of the gastrointestinal tract is important for cleaning gastrointestinal tract in preparation for the next meal. The impairment of the interdigestive contractile complex may be related to the dysmoility-like symptoms of W-mutant rats in vivo.
The ultrastructure of the wild-type (+/+) mice small intestine was compared with c-kit mutant (W/Wν) mice which only have few interstitial cells of Cajal (ICC) associated with Auerbach’s plexus, in order to elucidate whether the specialized membrane contacts are general features of so-called fibroblast-like cells that are widely distributed in the tunica muscularis of the alimentary tract. Fibroblast-like cells in the Auerbach region were found in approximately equal number in W/Wν mice as in +/+ mice, while ICC associated with Auerbach’s plexus (ICC-AP) could not be demonstrated in W/Wν mice in the present investigation. Fibroblast-like cells were characterized by cytoplasm of moderate to high electron density, well developed rough endoplasmic reticulum and nuclei with thick peripheral accumulations of heterochromatin. There were no basal lamina and caveolae along the cell membrane. It was observed that single fibroblast-like cells formed probable small gap junctions with muscle cells of both circular and longitudinal layers. Fibroblast-like cells with the same features were also observed in the region of the deep muscular plexus in both +/+ and W/Wν mice. The present observation, together with our previous studies on rats and guinea-pigs, suggest the common presence of gap junctions or gap junction-like structures on fibroblast-like cells in the gastrointestinal musculature and their involvement in the regulatory system of gastrointestinal motility by passing electrical or molecular signals to influence the state of muscle tonus.
Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Especially in this disease, qualitative and quantitative aspects render the dysregulated epidermal growth factor receptor (HER1/EGFR) an outstanding therapeutic target. A variety of therapeutic compounds was developed to target HER1/EGFR among which the clinically most advanced agents are small molecule tyrosine kinase (TK) inhibitors. Unfortunately, clinical studies examining their therapeutic efficacy have so far failed to document a major therapeutic break-through in the setting of GBM. Thus, the targeted approach against HER1/EGFR likely requires a synergistic drug combination strategy to ultimately become successful in this disease. This patents review focuses on innovative therapeutic strategies combining HER1/EGFR-targeted TK inhibitors with novel agents which for the most part have not been evaluated for the treatment of GBM yet but which constitute interesting candidates for further evaluation in this setting.
Studies in vitro have permitted the identification of enteric neural progenitor cells. Now the question arises as to where these progenitor cells are located in vivo. The purpose of this paper is to identify possible candidate cells by means of transmission electron microscopy (TEM). We have located three interstitial cellular types around the rat duodenum myenteric plexus. Type I cells have been identified as Interstitial Cells of Cajal (ICCs). These cells present well defined ultrastructural characteristics, including the triple connexion IC- nervous trunk- blood vessels. Type II cells show characteristics of immature cells, emphasizing the presence of a single cilium with the structure (9+0). To analyse this nanostructure, we have elaborated a reconstruction on ultrathin sections. The two previously described cellular types could be considered to be different functional states of the same cell. Type III cells present ultrastructural characteristics of fibroblast-like cells. This study suggests that Type II cells could be a source of neural progenitor cells.
To determine the expression of the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and c-Kit in vestibular schwannoma (VS) and to determine the potential role of imatinib mesylate (Gleevec) in regulating the growth and cell death of this tumor.
Protein tyrosine kinases are transmembrane tyrosine kinase receptors that transduce signals from inside and outside the cell and function as relay points for signaling pathways. They have a key role in numerous processes that affect cell proliferation, tumorigenesis, cancer invasion, metastasis, and modulation of apoptosis. A few of these kinases have been demonstrated to be overexpressed and dysregulated in many carcinomas, sarcomas, and benign tumors.
Immunohistochemical staining was used to investigate the expression of PDGFR and c-Kit in archived acoustic neuroma tissue. Clinical data including size of tumors, age, sex, and symptoms were correlated with kinase expression, whereas Western blot analysis and immunofluorescence were performed to demonstrate the expression and localization of PDGFR and c-Kit in HEI193, an immortalized VS cell line. Clonogenic survival assays were performed to assess proliferation inhibition by Gleevec. Gleevec's effect on the cell cycle profile also was investigated via flow cytometry analysis.
Expression of PDGFR in the formalin-fixed VS tumor tissue was observed in 23 (67.5%) of the 34 samples. C-kit was expressed in 18 (52.9%) of the 34 samples. Western blot analysis demonstrates positive expression of c-Kit and PDGFR-Q in HEI193 and a primary VS culture. Western blot analysis showed downregulation of phospho-c-kit and phospho-PDGFR-Q with 5 and 10 uM Gleevec. Immunofluorescent staining of this cell line also reveals that PDGFR-β is localized primarily in the cytoplasm, whereas c-Kit is both nuclear and cytoplasmic. Cell cycle analysis of HEI193 96 hours after incubation with Gleevec indicates a dose-dependent increase in G1 from 61.6% to 70.7% and 74% at 5 and 10 uM of Gleevec, respectively. Colony formation assays demonstrate dose-dependent growth inhibition by Gleevec, in the HEI193 cell line as well as in a VS cell culture derived from a fresh tumor.
The expression of PDGFR-Q and c-Kit in VS tissue may indicate novel molecular targets involved in the development of this tumor. Direct inhibition of these molecules by Gleevec may have relevant therapeutic applications.
In the gastrointestinal musculature, interstitial cells of Cajal (ICC) distribute and regulate the gastrointestinal motility. Another type of mesenchymal cell, known as the fibroblast-like cell (FLC), has also been reported to be juxtaposed to the ICC. In this study, we examined the immunohistochemical properties of FLC in the murine gastrointestinal musculature using antibodies to small conductance Ca(2+)-activated K(+) channel 3 (SK3), platelet-derived growth factor receptor alpha (PDGFRalpha), and CD34. SK3-immunopositive (SK3-ip) cells were observed in the musculature throughout the gastrointestinal tract. These SK3-ip cells were distinct from the ICC that were identified by c-Kit immunoreactivity. In the muscular layers, SK3-ip cells were bipolar in shape and were associated with the intramuscular ICC and nerve fiber bundles. In the myenteric layer multipolar-shaped SK3-ip cells encompassed the myenteric ganglia. SK3-ip cells in the subserosal plane formed a cellular network with their ramified processes. The distribution pattern of the SK3-ip cells in the ICC-deficient W(v)/W(v) mutant mice was similar to that in normal mice. We also demonstrated that SK3-ip cells showed the intense PDGFRalpha immunoreactivity that was previously examined in FLC. However, CD34 immunoreactivity, one of the markers of human FLC, was not observed in SK3-ip cells with the exception of subserosal FLC. Thus, our observations indicate that SK3- and PDGFRalpha-double immunopositive cells are FLC in the murine gastrointestinal musculature and behave as a basic cellular element throughout the gastrointestinal musculature.
Die Sekretion angiogener und hämatopoetischer Wachstumsfaktoren führt über autokrine und parakrine Signalübertragungsmechanismen zur Proliferation von Endothelzellen und leukämischen Blasten. Wachstumsfaktor-Rezeptoren stellen Zielstrukturen in der Krebstherapie dar. SU5416 ist ein niedermolekularer kompetitiver Phosphorylierungshemmer der Rezeptoren des Vaskulären Endothelialen Wachstumsfaktors (VEGFR-1 und -2) und des Stammzellfaktor-Rezeptors c-KIT. Wir zeigen die Behandlungsergebnisse mit SU5416 bei 7 Patienten mit akuten myeloischen Leukämien (AML). Bei zwei dieser Patienten (28%) konnte durch eine Monotherapie eine mehrmonatige Remission der Erkrankung erzielt werden, die anderen Patienten zeigten kein Ansprechen, was eine nur eingeschränkte Wirksamkeit in der Therapie der AML belegt. Weitere Studien mit dem Ziel der besseren Identifikation von Rezeptoren und der Prüfung von Kombinationen aus Rezeptortyrosinkinase-Inhibitoren und konventionellen Zytostatika sind erforderlich.
Interstitial cells of Cajal (ICC) are associated with afferent innervation and peristalsis of the stomach suggestive of a key role in the pathophysiology of gastroparesis. We studied changes in the density and ultrastructure of ICC and enteric nerves in the streptozotocin-induced diabetes mellitus (STZ-DM) in Wistar rats using immunohistochemistry and electron microscopy. Gastric emptying was studied in vivo by single-photon emission computed tomography. In the STZ-DM antrum, a marked reduction was observed in the density of the intramuscular ICC (ICC-IM) and ICC located at the submucosal border of the circular muscle layer of the antrum (ICC-SM). The surviving ICC showed lamellar bodies and partial vacuolation of the cytoplasm content, loss of connections between ICC-IM and nerves; it appeared that injured ICC-IM developed into fibroblast-like ICC. ICC associated with Auerbach's plexus (ICC-AP) in the antrum and ICC in the fundus were not affected significantly except for a loss of connections with nerve structures. Marked reduction in nerve tissue (Protein Gene Product-9.5 positivity) was also restricted to the muscle layers including nitrergic nerves (neuronal nitric oxide synthase positivity). In vivo assessed gastric emptying was markedly reduced in STZ-DM rats. Our data demonstrate in the STZ-DM rat stomach a decreased density of ICC limited to the antrum and to ICC-IM and ICC-SM, and structural degeneration in ICC-IM and associated nerves with a special emphasis on loss of synaptic connections, accompanied by a decrease in gastric emptying. Hence, in this model of gastroparetic diabetes, regional injury to subsets of ICC and nerves are associated with gastric motor dysfunction.
We report a case of vitiligo with localized repigmentation induced by imatinib mesylate. An elderly Chinese man has been characterized with recurrent gastrointestinal stromal tumors originated from interstitial cells of Cajal with gain-of-function c-kit mutation. His recurrent tumors have responded to imatinib mesylate therapy. To our surprise, his decade-long vitiligo was dramatically ameliorated by localized repigmentation, which is considered to be the side effect of imatinib mesylate. Hypopigmentation induced by imatinib mesylate with possible molecular blockage to a melanin-dependent KIT signal has been well documented; however, our case with repigmentation suggested that KIT signal of melanin formation would be much more sophisticated than we have believed.
The distribution of the c-kit receptor expressing cells and gap junction protein, connexin (Cx) 43 in the guinea-pig stomach (antrum), small intestine (jejunum) and colon (ascending) was studied by immunohistochemistry. The anti-c-kit protein immunopositive cells were regularly observed in the myenteric region throughout all three organs. The immunopositive cells were also sparsely distributed in the circular muscle layer of both the stomach and the colon, but not in the small intestine. They were densely located in the regions of the deep muscular plexus (DMP) of the small intestine and submuscular plexus (SMP) of the colon. In contrast, strong immunoreactivity to anti-Cx 43 antibody was observed in almost the entire thickness of the circular muscle layer of the stomach and the small intestine, but not in the colon. Dense immunoreaction deposits were observed in the region of the DMP and SMP. However, only very weak immunoreactivity to anti-Cx 43 antibody was detected in the myenteric region of all three organs. These results suggest that the c-kit receptor expressing cells or interstitial cells of Cajal (ICC) in the myenteric region of the three organs, and in the SMP of the colon, are poorly coupled with the bulk of circular muscle tissue by gap junctions, while ICC in the DMP and in the circular muscle layer of the stomach couple well with the surrounding muscle tissue.
Interstitial cells in the myenteric plexus and the deep muscular plexus of the small intestine of the c-kit mutant rats (Ws/Ws) and their normal siblings (+/+) were studied. c-Kit immunoreactivity was detected in two regions corresponding to the myenteric plexus and the deep muscular plexus in the jejunum of +/+ rats, while no immunoreactivity was detected in Ws/Ws rats. Using electron microscopy, two types of gap junction-forming interstitial cells were found in association with the myenteric plexus in +/+ rats: one type characterized by a typical fibroblastic ultrastructure, and the other characterized by numerous mitochondria and less electron-dense cytoplasm. Since the latter were greatly reduced in Ws/Ws rats, it was suggested that these cells correspond to c-kit-expressing cells, i.e. interstitial cells of Cajal in the myenteric plexus region. In contrast, two types of interstitial cells in the region of the deep muscular plexus were observed with no difference between +/+ and Ws/Ws rats. Probable interstitial cells of Cajal in this region were characterized by a basal lamina and numerous caveolae as well as large gap junctions that interconnect with each other and with the smooth muscle cells. We concluded that interstitial cells of Cajal in the rat intestine are heterogeneous in ultrastructure, c-kit dependency in the cell maturation, and functional role.
The tissue-specific expression of connexin subtypes in gap junctions between the interstitial cells and smooth muscle cells in the submuscular plexus of the colon has a functional importance in relation to intestinal pacemaker activity. Immunocytochemical observations of two types of connexin molecules, connexin43 and connexin45, were made with a confocal laser scanning microscope on cryosections of freshly frozen dog, guinea pig, mouse and rat proximal colon. Connexin43 immunoreactivity appeared as a series of dots along the submuscular plexus of guinea pig and dog. In contrast, connexin43 immunoreactivity was not found in mouse and rat colon. Connexin43 immunoreactivity was not observed in the colon muscular layer in the four animal species examined. In double-stained materials with a marker for either vimentin or smooth muscle actin, connexin43 immunoreactivity was colocalized with vimentin immunoreactivity, whereas it was not with either smooth muscle actin immunoreactivity or phalloidin reactivity. This indicated that the connexin43-expressing cells possess a vimentin-positive fibroblast-like nature rather than a smooth muscle-like one. In addition, in guinea pig colon, connexin43 immunoreactivity colocalized with c-Kit immunoreactivity. In conclusion, network-forming cells are connected by connexin43 gap junctions in the submuscular plexus of guinea pig and dog colon, most likely indicating that interstitial cells act as an intestinal pacemaker and conductive system.
Gestational maturation of gastrointestinal motility is a key factor in readiness of the preterm neonates for enteral nutrition. Since gastric motility mainly depends on the electrical activity of the smooth muscle cells, it was of interest to investigate the developmental aspects of electrical activity of the stomach. The latter was recorded weekly through cutaneous electrogastrography in 27 preterm infants (aged 29-34 weeks of gestation). Recordings were done for 1 hr before and 1 hr after meal. The electrogastrographic variables measured were: percentage of normal gastric rhythm, ie, 2-4 cpm; percentage of tachygastria (>4 cpm); the fed-to-fasting ratio of the dominant electrogastrographic power; and the instability coefficient of the dominant frequency. Data were compared with those measured in 10 full-term infants. Peaks of normal electrical activity (2-4 cpm) were present in most of the recordings at all the gestational ages; however, percentages of both normal electrical rhythm and tachygastria in preterm infants were similar to those measured in full-term infants (mean +/- SD) (normal rhythm; fasting: 70.2 +/- 3.8, fed: 72.2 +/- 5.0; tachygastria: fasting: 24.6 +/- 4.0, fed: 19.1 +/- 3.5) by 35 weeks of gestation (normal rhythm; fasting: 67.5 +/- 2.0, fed: 69.6 +/- 4.4; tachygastria: fasting: 27.1 +/- 4.0, fed: 25.6 +/- 4.1). The coefficient of instability of the dominant frequency in preterm infants was also similar to the value measured in full-term infants by 35 weeks of gestation, whereas the EGG power showed a significant increase in the postprandial state at all the gestational ages. We conclude that a maturation pattern of the electrical activity of the stomach can be detected by means of a noninvasive tool such as cutaneous electrogastrography: a normal electrical rhythm can be detected at very early gestational ages; however, this activity becomes dominant at around the 35 weeks of gestational age. In preterm infants developmental changes of gastric electrical activity are a function of advancing postnatal age.
Recent studies on the interstitial cells of Cajal (ICC) have determined ultrastructural criteria for the identification of these previously enigmatic cells. This review deals with the electron microscopic findings obtained by the author's research group in different tissue regions of the gut in mice, rats and guinea-pigs, comparing these with reports from other groups in different species and in humans. ICC are characterized by the following morphological criteria: numerous mitochondria, abundant intermediate filaments and large gap junctions which connect the cells with each other and with smooth muscle cells. Due to their location in the gut and the specific species, the ICC are markedly heterogeneous in appearance, ranging from cells closely resembling smooth muscle cells to those similar to fibroblasts (Table 1). Nevertheless, the above-mentioned morphological features are shared by all types of ICC and serve in identifying them. Recent discoveries on a significant role of c- kit in the maturation of the ICC and their specific immunoreactivity to anti-c-Kit antibody have confirmed the view that the ICC comprise an independent and specific entity of cells. This view is reinforced by the findings of the author's group that the ICC characteristically possess vimentin filaments and are stained with the zinc iodide-osmium tetroxide method which provides a staining affinity similar to methylene blue, the dye used in the original work by Cajal, (1911). Developmental studies indicate that the ICC are derived from a non-neuronal, mesenchymal origin. This paper further reviews advances in the physiological studies on the ICC, in support of the hypothesis by THUNEBERG (1982) that they function as a pacemaker in the digestive tract and a mediator transmitting impulses from the nerve terminals to the smooth muscle cells.
This paper reviews the distribution of interstitial cells of Cajal (ICC) in the human gastrointestinal (GI) tract, based on ultrastructural and immunohistochemical evidence. The distribution and morphology of ICC at each level of the normal GI tracts is addressed from the perspective of their functional significance. Alterations of ICC reported in achalasia of cardia, infantile hypertrophic pyloric stenosis, chronic intestinal pseudoobstruction, Hirschsprung's disease, inflammatory bowel diseases, slow transit constipation, and some other disorders of GI motility as well as in gastrointestinal stromal tumors are reviewed, with emphasis on the place of ICC in the pathophysiology of disease.
The shape, distribution, and ultrastructural features of interstitial cells of Cajal (ICC) of different tissue layers and organs of the rat and guinea-pig digestive tract were described and compared with the corresponding cells in other species including mice, dogs, and humans, as reported in the literature. By light microscopy, the best marker for ICC appeared to be immunoreactivity for c-Kit. Ultrastructurally, ICC were characterized by the presence of many mitochondria, bundles of intermediate filaments, and gap junctions, which linked ICC with each other. However, ICC were morphologically heterogeneous and had particular features, depending on their tissue and organ location and species. ICC in the deep muscular plexus of the small intestine and in the submuscular plexus of the colon were the most like smooth muscle cells, and had a distinct basal lamina and numerous caveolae. In contrast, ICC of Auerbach's plexus at all levels of the gastrointestinal tract were the least like smooth muscle cells. They most closely resembled unremarkable fibroblasts. ICC within the circular muscle layer were intermediate in form. In addition to the tissue specificity, some organ and species specificity could be distinguished. The structural differences between ICC may be determined by their microenvironment, including the effects of mechanical force, type of nerve supply, and spacial relationship with smooth muscle cells.
The interstitial cell of Cajal, abbreviated ICC, is a specific cell type with a characteristic distribution in the smooth muscle wall throughout the alimentary tract in humans and laboratory mammals. The number of publications relating to ICC is rapidly increasing and demonstrate a rich variation in the structure and organization of these cells. This variation is species-, region-, and location-dependent. We have chosen to define a "reference ICC," basically the ICC in the murine small intestine, as a platform for discussion of variability. The growing field of ICC markers for light and electron microscopy is reviewed. Although there is a rapidly increasing number of approaches applicable to bright field and fluorescence microscopy, the location of markers by electron microscopy still suffers from inadequate preservation of ultrastructural detail. Finally, we summarize evidence related to ICC ultrastructure under conditions differing from those of the normal, adult individual (during differentiation, in pathological conditions, transplants, mutants, and in cell culture).
The localization of the neurokinin 1 receptor in rat and guinea pig gastrointestinal tract has been extensively studied but not in human tissues. The present study used antibodies to characterize the cellular expression of neurokinin 1 receptors in human antrum. Cryostat sections (40-80 microm) were immunostained for the neurokinin 1 receptor double labeled with substance P, von Willebrand's factor, c-kit, fibronectin, S-100, serotonin, gastrin and somatostatin. Neurokinin 1 receptor-immunoreactivity was observed on neurons within the myenteric and submucosal plexuses surrounded by substance P-immunoreactive fibers and on von Willebrand's factor-immunoreactive endothelial cells lining blood vessels throughout the antral wall. c-Kit-immunoreactive interstitial cells of Cajal and gastrin cells were co-stained by the monoclonal neurokinin 1 receptor antibody. Finally, there was no evidence for the presence of the neurokinin 1 receptor on fibroblasts, Schwann, somatostatin, serotonin or smooth muscle cells. This study clearly demonstrates an expanded cellular expression of the neurokinin 1 receptor in the human antrum.
STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.
STI571 (formerly known as CGP 57148B) is a protein-tyrosine kinase inhibitor that is currently in clinical trials for the treatment of chronic myelogenous leukemia. STI571 selectively inhibits the Abl and platelet-derived growth factor (PDGF) receptor tyrosine kinases in vitro and blocks cellular proliferation and tumor growth of Bcr-abl- or v-abl-expressing cells. We have further investigated the profile of STI571 against related receptor tyrosine kinases. STI571 was found to potently inhibit the kinase activity of the alpha- and beta-PDGF receptors and the receptor for stem cell factor, but not the closely related c-Fms, Flt-3, Kdr, Flt-1, and Tek tyrosine kinases. Additionally, no inhibition of c-Met or nonreceptor tyrosine kinases such as Src and Jak-2 has been observed. In cell-based assays, STI571 selectively inhibited PDGF and stem cell factor-mediated cellular signaling, including ligand-stimulated receptor autophosphorylation, inositol phosphate formation, and mitogen-activated protein kinase activation and proliferation. These results expand the profile of STI571 and suggest that in addition to chronic myelogenous leukemia, STI571 may have clinical potential in the treatment of diseases that involve abnormal activation of c-Kit or PDGF receptor tyrosine kinases.
A discovery that the protooncogene encoding the receptor tyrosine kinase, c-kit, is allelic with the Dominant white spotting (W) locus establishes that c-kit plays a functional role in the development of three cell lineages, melanocyte, germ cell, and hematopoietic cell which are defective in W mutant mice. Recent analyses of c-kit expression in various tissues of mouse, however, have demonstrated that c-kit is expressed in more diverse tissues which are phenotypically normal in W mutant mice. Thus, whether or not c-kit expressed outside the three known cell lineages plays a functional role is one of the important questions needing answering in order to fully elucidate the role of c-kit in the development of the mouse. Here, we report that some of the cells in smooth muscle layers of developing intestine express c-kit. Blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (mAb) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralytic ileus. Physiological analysis indicates that the mechanisms required for the autonomic pacing of contraction in an isolated gut segment are defective in the anti-c-kit mAb-treated mice, W/Wv mice and even W/+ mice. These findings suggest that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of autonomic gut motility.
The ultrastructure and immunocytochemistry of the canine distal pyloric muscle loop, the pyloric sphincter, were studied. Cells in this muscle were connected by gap junctions, fewer than in the antrum or corpus. The sphincter had a dense innervation and a sparse population of interstitial cells of Cajal. Most such cells were of the circular muscle type but a few were of the type in the myenteric plexus. Nerves were sometimes associated with interstitial cell profiles, but most nerves were neither close to nor associated with interstitial cells nor close to smooth muscle cells. Nerve profiles were characterized by an unusually high proportion of varicosities with a majority or a high proportion of large granular vesicles. Many of these were shown to contain material immunoreactive for vasoactive intestinal polypeptide (VIP) and some had substance P (SP) immunoreactive material. All were presumed to be peptidergic. VIP was present in a higher concentration in this muscle than in adjacent antral or duodenal circular muscle. Interstitial cells of Cajal made gap junctions to smooth muscle and to one another and might provide myogenic pacemaking activity for this muscle, but there was no evidence of a close or special relationship between nerves with VIP or SP and these cells. The absence of close relationships between nerves and either interstitial cells or smooth muscle cells leaves unanswered questions about the structural basis for previous observations of discrete excitatory responses or pyloric sphincter to single stimuli or nerves up to one per second. In conclusion, the structural observations suggest that this muscle has special neural and myogenic control systems and that interstitial cells may function to control myogenic activity of this muscle but not to mediate neural signals.
The structural relationship of nerve, muscle, and interstitial cells of Cajal in circular muscle of the lesser curvature of the dog stomach (corpus) has been studied. This muscle has also been characterized functionally. Muscle cells are arranged in bundles and are interconnected by numerous gap junctions averaging 30 per 100 cross-sectioned muscle cells, and leading to an estimate that each cell has about 200 gap junctions. No other smooth muscle studied to date has such a high density of gap junctions. Nerve varicosities, mostly containing a predominance of small agranular vesicles with some containing a predominance of large granular vesicles, are located outside muscle bundles, usually in small- to medium-sized bundles. Very few nerves containing small granular vesicles, presumably adrenergic, were found in agreement with functional studies. A substantial number of damaged nerve profiles was also found, perhaps contributing to the loss of nerve-dependent responses present in vivo, but absent in vitro. Interstitial cells of Cajal were rare in this tissue, about 1 per 1000 cross-sectioned muscle cells. When present, they often made gap junction contact with smooth muscle and were closely innervated. The findings of a structural basis for very tight coupling between cells, the absence of a structural basis for direct neural control over motor function, and other findings have implications for the control of contractions in this muscle.
The pacemaker activity in the mammalian gut is responsible for generating anally propagating phasic contractions. The cellular basis for this intrinsic activity is unknown. The smooth muscle cells of the external muscle layers and the innervated cellular network of interstitial cells of Cajal, which is closely associated with the external muscle layers of the mammalian gut, have both been proposed to stimulate pacemaker activity. The interstitial cells of Cajal were identified in the last century but their developmental origin and function have remained unclear. Here we show that the interstitial cells of Cajal express the Kit receptor tyrosine kinase. Furthermore, mice with mutations in the dominant white spotting (W) locus, which have cellular defects in haematopoiesis, melanogenesis and gametogenesis as a result of mutations in the Kit gene, also lack the network of interstitial cells of Cajal associated with Auerbach's nerve plexus and intestinal pacemaker activity.
The ultrastructure of plexus muscularis profundus (PMP) of the mouse small intestine was investigated subsequent to vascular perfusion with ruthenium red-containing and routine aldehyde fixatives. Four types of nerve terminals were revealed. Type I: numerous 500-Å agranular vesicles and few 1,000-Å granular vesicles. Type II: predominantly large (1,000–1,500 Å), granular vesicles and fewer 500-Å agranular vesicles. Type III: an abundance of mitochondria and many flattened vesicles (300 Å × 700-1,300 Å). Type IV was identified by abundant smooth cisternae 200 Å in width. Types I-III formed close (200 Å), synapselike contacts to interstitial cells of Cajal (ICC-III). Presynaptic densities were frequent in type I endings. A direct innervation of muscle cells via PMP was only very occasionally suggested. ICC-III possessed a basal lamina and numerous caveolae associated with subsurface SER-cisternae. Mitochondria were very abundant in ICC-III-processes. ICC-III formed multiple, large gap junctions with outer circular-muscle cells and with other ICC-III. Also reflexive gap junctions were observed. Fibroblastlike cells (FLC) were distinguished by their prominent GER, the frequent presence of lipid droplets, and the lack of caveolae and a basal lamina. FLC never participated in synaptic arrangements or gap junctions. Macrophagelike cells were occasionally encountered. It is concluded that possible efferent and afferent nerve terminals in PMP may chiefly, if not exclusively, innervate ICC-III, the ultrastructure of which is compatible with efferent and/or afferent modulatory actions.
Systematic examination using electron microscopic montages and serial sections has demonstrated that three types of interstitial cell, namely gap junction-rich cells, glycogen-rich cells and fibroblast-like cells, are densely located along the whole extent of the deep muscular plexus of the guinea pig small intestine. They tend to be distributed in an alternating fashion in the cellular network, connected with muscle cells of the outer, circular layer by means of gap junctions. These three types of interstitial cell show close relations to two types of nerve varicosity: one type is characterized by clear round vesicles with diameters of about 50 nm, and the other by flattened vesicles measuring about 35 nm by 70 nm. Electron-dense patches have been observed at the cytoplasmic side of the axonal membranes. Muscle cells of both inner and outer circular layers also show close relations to these two types of nerve varicosity. These morphological features are discussed with the implication that they may have some regulatory role in intestinal movement.
Evidence showing that interstitial cells of Cajal have important regulatory functions in the gut musculature is accumulating. In the current study, the ultrastructure of the deep muscular plexus and associated interstial cells of Cajal in human small intestine were studied to provide a reference for identification and further physiological or pathological studies. The deep muscular plexus was sandwiched between a thin inner layer of smooth muscle (one to five cells thick) and the bulk of the circular muscle. Interstitial cells of Cajal in this region very much resembled smooth muscle cells (with a continuous basal lamina, caveolae, intermediate filaments, dense bodies, dense bands, and a well-developed subsurface smooth endoplasmic reticulum), but the arrangement of organelles was clearly different, and cisternae of granular endoplasmic reticulum were abundant. Interstitial cells of Cajal were distinguished from fibroblasts or macrophages in the region. They ramified in the inner zone of the outer division of circular muscle, penetrated the inner-most circular layer, and were also found at the submucosal border. They were in close, synapselike contact with nerve terminals of the deep muscular plexus, and only few gap junctions with other interstitial cells of Cajal or with the musculature were observed. Compared with interstitial cells of Cajal from other mammals, those associated with the deep muscular plexus in the human small intestine more closely resemble smooth muscle cells, and their organization appears more diffuse; however, the ultrastructure and organization of interstitial cells of Cajal is compatible with modulatory actions on the circular muscle also in humans.
Homozygous mutant rats at the newly found white spotting (Ws) locus were anemic and deficient in mast cells and melanocytes. Because the phenotype of Ws/Ws rats resembled the phenotype of mice possessing a double-gene dose of mutant alleles at the W locus and because the c-kit gene was mapped at the W locus of mice, we characterized the c-kit gene of Ws/Ws rats. The authentic sequence of the rat c-kit cDNA was determined by using a cDNA library prepared from the hippocampus of Sprague-Dawley rats. The c-kit cDNA of Ws/Ws and normal (+/+) control rats was obtained by reverse transcriptase modification of the polymerase chain reaction. When compared with the authentic sequence, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats. This change was shown to be a result of the deletion of the genomic DNA. Four amino acids encoded by the deleted 12 bases (ie, Val-Lys-Gly-Asn) were located at two amino acids downstream from the tyrosine autophosphorylation site in the c-kit kinase and were conserved not only in mouse and human c-kit kinases but also in mouse and human c-fms kinases (ie, receptors of colony-stimulating factor-1). Taken together, the Ws/Ws rat is the first characterized mutant of the c-kit gene in an animal species other than the mouse.
Mice carrying mutations at the W locus located on chromosome 5 are characterized by severe macrocytic anaemia, lack of hair pigmentation and sterility. Mutations at this locus appear to affect the proliferation and/or migration of cells during early embryogenesis and result in an intrinsic defect in the haematopoietic stem cell hierarchy. An understanding of the molecular basis of the complex and pleiotropic phenotype in W mutant mice would thus provide insights into the important developmental processes of gametogenesis, melanogenesis and haematopoiesis. Here we show that the mouse mutant W has a deletion of the c-kit proto-oncogene. Interspecific backcross analysis demonstrates that the W locus is very tightly linked to c-kit and that the two loci cannot be segregated at this level of analysis. c-kit is the cellular homologue of the oncogene v-kit of the HZ4 feline sarcoma virus and encodes a transmembrane protein tyrosine kinase receptor that is structurally similar to the receptors for colony-stimulating factor-1 (CSF-1) and platelet derived growth factor. The co-localization of c-kit with W provides a molecular entry into this important region of the mouse genome. In addition, these observations provide the first example of a germ-line mutation in a mammalian proto-oncogene and implicate the c-kit gene as a candidate for the W locus.
Mutations at the W locus in the mouse have pleiotropic effects on embryonic development and hematopoiesis. The characteristic phenotype of mutants at this locus, which includes white coat color, sterility, and anemia, can be attributed to the failure of stem cell populations to migrate and/or proliferate effectively during development. Mapping experiments suggest that the c-kit proto-oncogene, which encodes a putative tyrosine kinase receptor, is a candidate for the W locus. We show here that the c-kit gene is disrupted in two spontaneous mutant W alleles, W44 and Wx. Genomic DNA that encodes amino acids 240 to 342 of the c-kit polypeptide is disrupted in W44; the region encoding amino acids 342 to 791 is disrupted in Wx. W44 homozygotes exhibit a marked reduction in levels of c-kit mRNA. These results strongly support the identification of c-kit as the gene product of the W locus.
The ultrastructure of the region shown to be essential for pacemaking activity of the circular muscle of the canine colon was studied. This region, at the inner border of the circular muscle, consists of a network of several layers of interstitial cells of Cajal type III. These are interconnected to one another and to the adjacent circular muscle cells by numerous gap junctions. Elsewhere in circular muscle, gap junctions are rare and small. In addition, interstitial cells are in close (often less than 20 nm) contact with nerve varicosities containing large granular vesicles or sometimes small granular vesicles. The morphology of interstitial cells resembles that of others of type III. It is suggested that this arrangement of interstitial cells, circular smooth muscles, and nerves allows for a tightly coupled network of membrane oscillators to be subject to neural modulation.
Interstitial cells of Cajal (ICCs) are believed to initiate the basic contractile activity of the gastrointestinal tract. Because ICCs in the intestine of mice express c-kit receptor tyrosine kinase and because rats are more commonly used than mice for pathophysiological investigations of the gastrointestinal tract, the number of the c-kit messenger RNA-expressing cells was compared with gastrointestinal movement in rats.
The c-kit messenger RNA-expressing cells were detected by in situ hybridization. The autonomous contraction of excised segments of the ileum was recorded. The function of the pyloric sphincter was evaluated by measuring the content of bile acids in the stomach.
The c-kit messenger RNA-expressing cells were not detectable in the stomach of Ws/Ws mutant rats with a small deletion at the tyrosine kinase domain of c-kit, and the number of c-kit messenger RNA-expressing cells decreased to 7% that of normal control rats in the ileum of Ws/Ws rats. The contractile activity of the ileum was apparently impaired, and the content of bile acids in the stomach was significantly increased in Ws/Ws rats.
The abnormalities in the ileal movement and pyloric sphincter function in Ws/Ws rats were attributable to the deficiency of c-kit messenger RNA-expressing cells.
1. Interstitial cells of Cajal (ICs) have been proposed as pacemakers in the gastrointestinal tract. We studied the characteristics and distribution of ICs and electrical activity of small intestinal muscles from mice with mutations at the dominant-white spotting/c-kit (W) locus because the tyrosine kinase function of c-kit may be important in the development of the IC network. 2. W/WV mutants (days 3-30 postpartum) had few ICs in the myenteric plexus region compared with wild type (+/+) siblings. The few ICs present were associated with neural elements and lay between myenteric ganglia and the longitudinal muscle layer. 3. Electrical recordings from intestinal muscle strips showed that electrical slow waves were always present in muscles of +/+ siblings, but were absent in W/WV mice. 4. Muscles from W/WV mice responded to stimulation of intrinsic nerves. Neural responses, attributed to the release of acetylcholine, nitric oxide and other unidentified transmitters, were recorded. 5. These findings are consistent with the hypothesis that ICs are a critical element in the generation of electrical rhythmicity in intestinal muscles. The data also show that neural regulation of gastrointestinal muscles can develop independently of the IC network. 6. W locus mutants provide a powerful new model for studies of the physiological role of ICs and the significance of electrical rhythmicity to normal gastrointestinal motility.
The network of interstitial cells of Cajal (ICC) at the submucosal surface of the canine colon was selectively stained by incubation with 15-50 microM methylene blue for 30-45 min. The network was composed of regularly scattered ICC cell bodies interconnected by long processes. Circular muscle cells were unstained. Staining of neurons was limited to one or two axons within bundles. The ICC network had a thickness of a single cell, since no overlapping of ICC cell bodies was observed. The ICC network connected the circular muscle cells at the submucosal surface across the septa which circumferentially divided the circular muscle into lamellae. Methylene blue at 50 microM slightly decreased the resting membrane potential and increased the duration of slow waves, leading to an increase in the force of phasic contractions, with no significant influence on other slow-wave parameters. Methylene blue produced neither electrophysiological nor mechanical effects on circular muscle preparations from which the submuscular ICC network was removed, indicating that the excitatory effects of methylene blue on the full-thickness circular muscle layer were mediated by ICC. In summary, the three-dimensional aspects of the submuscular ICC network can be visualized after selective staining by methylene blue. This staining does not affect physiological characteristics of smooth muscle cells.
It has been suggested that interstitial cells of Cajal (ICC) at the submucosal border of the colonic circular muscle are pacemaker cells. We studied smooth muscle cells and ICC at the submucosal surface of the circular muscle layer of the normal human colon.
Resected, unaffected specimens from human colon were studied by light microscopy and transmission electron microscopy.
Throughout the colon, the inner fourth of each circular-muscle lamella was covered with a layer of 2 to 15 muscle cells (ICMC) with a smaller diameter, more perinuclear organelles, and a greater glycogen content than the outer circular muscle cells. ICMC were interconnected by adherens junctions and close appositions. Small bundles of ICMC were present in the submucosa. ICC were identified in all regions of the colon (ascendens, transversum, and sigmoideum) at the submucosal border, in deeper parts of the submucosa in close contact with smooth muscle bundles as well as in the circular muscle and main septa. ICC had a continuous basal lamina, caveolae, dense bands, thin and intermediate filaments, dense bodies and a well-developed smooth endoplasmic reticulum. Mitochondria and granular endoplasmic reticulum were very abundant. Lipid droplets and glycogen granules were frequent. Thick (myosin) filaments were absent. Close contacts to nerves and gap junctions to other ICC or smooth muscle cells were exceptional. Fibroblast-like cells in the submucosa were rich in granular endoplasmic reticulum and intermediate filaments. They had few dense bands and caveolae. Mitochondria, smooth cisternae and glycogen granules were sparse, cytoplasmic dense bodies and a continuous basal lamina were lacking. Fibroblast-like cells were associated closely with collagen bundles and they had no close contacts with nerves, ICC or muscle cells.
Throughout the normal human colon, submucosal ICC and ICMC are identified and distinguished from other cell types present. Their organization and cytology differ from that of other animal species. The ultrastructure of ICC and ICMC is compatible with important regulatory functions on the circular muscle in the entire human colon.
Observation of whole mount stretch preparations using the zinc-iodide-osmic acid method reveals a wide variety of interstitial cells in different tissue layers of the guinea-pig small intestine. And a subsequent electron-microscopic examination and survey of references makes clear that the interstitial cells of Cajal (ICC) depicted in original drawings of Cajal are heterogeneous and correspond to different types of interstitial cells. The myenteric ICC are characterized by long dichotomous branching processes which constitute cellular networks independent from the nerve plexus and form many gap junctions at their tips. Their ultrastructure is similar to that of fibroblasts and they have no basal lamina. The myenteric ICC show strong immunoreactivity for vimentin and the c-kit receptor, and probably correspond to the intestinal pacemaker cells. Within the circular muscle layer, ICC are represented by the cells that are closely associated with fine nerve bundles. The ICC have various shapes, ranging from bipolar to stellate, depending on the running pattern of the nerve fibers that they are associated with. They show fibroblast-like ultrastructure and have no basal lamina. They form gap junctions with smooth muscle cells and are immunoreactive for vimentin. On the other hand, ICC associated with the deep muscular plexus described in the guinea-pig by Cajal could not be clearly identified. However, it is suggested that the ICC in this location may correspond to glycogen-rich cells possessing a basal lamina. Although they show a fairly well-developed rough endoplasmic reticulum, Golgi apparatus and immunoreactivity for vimentin, ICC of the deep muscular plexus are probably specialized smooth muscle cells in nature.
In-terstitial cells in deep muscular plexus of canine small intes-tine may be specialized smooth muscle cells) c-kit-Dependent development of interstiti-al cells and electrical activity in the murine gastrointestinal tract
- S S Kobayashi
- Gerthoffer
- Wt
- – Sanders
- S Torihashi
- Ward
- Sm
- S-I Nishikawa
- K Nishi
- S Kobayashi
- Sanders
- Km
S, Kobayashi S, Gerthoffer WT, Sanders KM (1993) In-terstitial cells in deep muscular plexus of canine small intes-tine may be specialized smooth muscle cells. Am J Physiol 265:G638–G645 Torihashi S, Ward SM, Nishikawa S-I, Nishi K, Kobayashi S, Sanders KM (1995) c-kit-Dependent development of interstiti-al cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 280:97–111
Morphology of the canine pyloric sphincter in relation to function An ul-trastructural study of the interstitial cells of Cajal of the hu-man stomach
- Ee
- I Berezin
- Allescher Hd
- H Manaka
- Daniel V Faussone-Pellegrini Posey
- Ms
- D Pantalone
- C Cortesini
EE, Berezin I, Allescher HD, Manaka H, Posey-Daniel V (1989) Morphology of the canine pyloric sphincter in relation to function. Can J Physiol Pharmacol 67:1560–1573 Faussone-Pellegrini MS, Pantalone D, Cortesini C (1989) An ul-trastructural study of the interstitial cells of Cajal of the hu-man stomach. J Submicrosc Cytol Pathol 21:439–460
Impaired development of interstitial cells and intestinal elec-trical rhythmicity in steel mutants Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine, with special reference to the interstitial cells of Cajal
- Sm
- Burns Aj
- S Torihashi
- Harney
- Sc
- Sanders
- Km
SM, Burns AJ, Torihashi S, Harney SC, Sanders KM (1995) Impaired development of interstitial cells and intestinal elec-trical rhythmicity in steel mutants. Am J Physiol 269: C1577–C1585 Zhou DS, Komuro T (1992a) Interstitial cells associated with the deep muscular plexus of the guinea-pig small intestine, with special reference to the interstitial cells of Cajal. Cell Tissue Res 268:205–216